The methods for identification and characterization of TAA, which represent the starting point for a tumor vaccine, are—on the one hand—based on the use of patient derived CTL or antibodies. This immunological approach is combined either with a gene expression approach or with a mass-spectrometry (MS)-assisted sequencing of the recognized peptides (see van der Bruggen, et al., 1991, A gene encoding an antigen recognized by cytolytic Tlymphocytes on a human melanoma, Science 254: 1643-1647, and Cox et al., 1994, Identification of a peptide recognized by five melanoma-specific human cytotoxic T-cell lines, Science 264: 716-719). Methods for identifying TAA which are based on comparative transcription profiling of tumorous and corresponding normal tissue are—for example—hybridization and the use of DNA microarray technology.
Celis et al., 1994, Induction of anti-tumor cytotoxic T-lymphocytes in normal humans using primary cultures and synthetic peptide epitopes, Proc. Natl. Acad. Sci. USA 91: 2105-2109, applied a method which takes advantage of the prediction of MHC class I-ligands derived from a selected tumor-associated antigen, and in which these ligands were verified as Tcell epitopes in a next step.
Drawbacks of patients-derived T-cell-based approaches are the extensive culture techniques and their restriction to the frequency of pre-existing T-cells.
Another T-cell-independent approach also known in the art combines epitope prediction and screening for predicted peptides in complex peptide mixtures, the peptides being identified by highly sensitive capillary liquid chromatography mass spectrometry (LC-MS) (see Schirle et al., 2000, Identification of tumor-associated MHC class I-ligands by a novel T-cell independent approach, Eur. J. Immunol. 30: 2216-2225).
DNA microarray technology provides a new approach using comparative expression profiling of tumorous and corresponding autologous normal tissue. Young et al., 2001, Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers, Am. J. Pathol. 158: 1639-1651, disclosed that by using this technique a large number of tumor-associated antigens from individual tumor samples can be identified. MHC-I-ligands derived from overexpressed or selectively expressed proteins provide possible targets for specific CTL recognition of tumors. Mathiassen et al., 2001, Tumor-associated antigens identified by mRNA expression profiling induce protective antitumor immunity, Eur. J. Immuno. 31: 1239-1246, demonstrated, in a mouse model, that by combining expression analysis with epitope prediction a successful vaccine can be prepared.
However, a disadvantage is that epitope prediction even for only a few target genes results in the identification of a vast number of candidate peptides, the majority of which are actually not presented by MHC molecules and thus do not induce a CTL-response.